Tissue engineered substitutes have the potential to provide effective, functional replacements of failing tissues and organs and as such help resolve the current transplantation crisis. To realize its potential, tissue engineering must generate substitutes that can be preserved in the long-term. Preservation is essential for off-the-shelf availability, storage and distribution of constructs fabricated at a large-scale at centralized locations, as well as for sterlity testing and quality control. The long-range goal associated with this research program is to develop a fundamental understanding of how the various cryopreservation parameters affect cellular and tissue construct function, and on the basis of this knowledge to develop widely applicable preservation protocols. The objective of this application is to evaluate the effect of ice-forming and ice-free cryopreservation on cell viability and construct function for a model pancreatic tissue substitute consisting of insulin-secreting cells and a hydrogel biomaterial. The central hypothesis is that entire tissue engineered substitutes can be successfully vitrified, while maintaining key structural and functional features of the cellular and biomaterial components;vitrification produces superior outcomes relative to conventional, ice-forming cryopreservation in terms of both construct integrity and functionality. Guided by strong preliminary data, the hypothesis will be addressed by the following 3 specific aims: 1) determine cell osmotic tolerance limits and cryoprotectant cytotoxicity and define the domain of conditions under which to cryopreserve cells used in tissue substitutes;2) characterize in vitro the effect of cryopreservation on construct structure and function;3) evaluate the in vivo functionality of cryopreserved substitutes. The effect of cryopreservation on cells will be assessed on the basis of cell viability, apoptosis, stress protein expression, and metabolic and secretory functions;and on biomaterials on the basis of their structural integrity and functionality. The approach is innovative, as it focuses on studying the effects of cryopreservation on both the cellular and biomaterial components of a three-dimensional (3D) tissue substitute. The proposed research is significant, as it will generate new fundamental information on the science of cryopreservation, provide a quantitative framework for the rational design of cryopreservation protocols, and identify key design features of tissue substitutes that enable their cryopreservation.